Microtubules (MTs) form complex intracellular networks that not only provide mechanical stability for cell structure and motility, but also from the tracks along which molecular motors traffic cargos to specific sub- cellular sites. While most MT networks have a relatively short half-life, recent work has shown that a subset (-10%) of post-translationally modified MTs are highly stable and function in a various biological processes. However, their role and regulation during viral infection remains poorly understood. Our screens for host factors that determine cellular susceptibility to infection by various viruses, in collaboration with Gundersen (Project 1), Walsh (Project 3) and Goff (Project 4) identified a number of novel regulators of stable MT formation, highlighting the potential role of this stable subset of MTs as specialized tracks for viral trafficking. Our collaborative effort with Gundersen to explore this more directly resulted in identification of the kinesin, Kif4 as an EBl-interacting regulator of MT stability and HIV-1 infection. In addition, Kif4 is also bound by Gag, a retroviral structural polyprotein that has been suggested to promote MT stabilization. Combined with our preliminary data showing that HIV-1 infection promotes stable MT formation and incoming viral cores colocalize with this stable MT subset, this suggests that Kif4:EB1 complexes represent a specific MT tip- binding complex specifically targeted by HIV-1. In this proposal we will map the domains in Kif4 and EBl that mediate their effects on stability and trafficking of HlV-1 during both early stage (inward-directed) movement of viral cores and late stage (outward-directed) movement and budding of viral particles. We will also determine the effects of infection or Gag expression on Kif4:EB1 complex formation and function in mediating MT stabilization during distinct phases of infection, and define the domains in Gag that mediate interactions with Kif4:EB1 complexes to determine how this viral protein affects complex formation and/or activity. Finally, we will determine whether infection or Gag expression manipulates localized signaling nodes to regulate MT formation and/or Kif4:EB1 function as virions enter the cell. This work takes advantage of working closely with members of this PPG with expertise in cytoskeletal regulation, motor-based virion movement, live imaging and cell signaling, as well as using the Imaging Core to develop a real-time image of the dynamic events involved in viral interactions with host MT networks and the role of highly specialized MT regulators in these events. This will provide detailed mechanistic insight into the role of MT regulators and subsets during HIV-1 infection, with important implications for our general understanding of MT regulation and cargo transport.